Novel crispr enzymes and systems
OwnerFeng Zhang, Bernd Zetsche +2inventors
InventorsFeng Zhang, Bernd Zetsche, Matthias Heidenreich, Sourav Choudhury
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Abstract
The invention provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides non-naturally occurring or engineered DNA or RNA-targeting systems comprising a novel DNA or RNA-targeting CRISPR effector protein and at least one targeting nucleic acid component like a guide RNA.
Claims66
An engineered, non-naturally occurring Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR)-CRISPR associated (Cas) (CRISPR-Cas) system comprising
- a) one or more Type V CRISPR-Cas polynucleotide sequences comprising a guide RNA which comprises a guide sequence linked to a direct repeat sequence, wherein the guide sequence is capable of hybridizing with a target sequence, or one or more nucleotide sequences encoding the one or more Type V CRISPR-Cas polynucleotide sequences, and
- b) a Cpf1 effector protein, or one or more nucleotide sequences encoding the Cpf1 effector protein;
- wherein the one or more guide sequences hybridize to said target sequence, said target sequence is 3′ of a Protospacer Adjacent Motif (PAM), and said guide RNA forms a complex with the Cpf1 effector protein;
- wherein the Cpf1 effector protein has at least 90% sequence identity with the Cpf1 effector protein from, Moraxella bovoculi AAX08_00205 or Moraxella bovoculi AAX11_00205.
The method of claim 15, wherein the target locus of interest is within a cell.
The method of claim 16, wherein the cell is a eukaryotic cell.
The method of claim 22, wherein the DNA is relaxed or supercoiled.
The method of claim 24, wherein the single nucleic acid component comprises a guide sequence linked to a direct repeat sequence.
The method of claim 26, wherein the strand break comprises a staggered DNA double stranded break with a 4 or 5-nt 5′ overhang.
The method of claim 33 wherein the particles comprise a lipid, a sugar, a metal or a protein.
A cell from the method of claim 37, or progeny thereof, wherein the cell comprises a modification not present in a cell not subjected to the method.
The cell of claim 38, of progeny thereof, wherein the cell not subjected to the method comprises an abnormality and the cell from the method has the abnormality addressed or corrected.
The cell product of claim 40, wherein the cell not subjected to the method comprises an abnormality and the cell product reflects the abnormality having been addressed or corrected by the method.
The host cell or cell line or progeny thereof according to claim 42, wherein the cell is a eukaryotic cell.
The host cell or cell line or progeny thereof according to claim 43, wherein the cell is an animal cell.
The method of claim 49, further comprising introducing the identified gene of interest into a plant cell or plant cell line or plant germplasm and generating a plant therefrom, whereby the plant contains the gene of interest.
The method of claim 50 wherein the plant exhibits the trait of interest.
The particle of claim 52, wherein the particle contains the Cpf1 effector protein complexed with the guide RNA.
The system or method of claim 56, wherein the cytidine deaminase is fused to the carboxy terminus of the Cpf1 effector protein.
The system or method of claim 56 or 57, wherein the Cpf1 effector protein or the cytidine deaminase is further fused to a uracil DNA glycosylase inhibitor.
The system or method of any of claims 56-58, wherein the Cpf1 effector protein comprises a catalytically inactive Nuc domain.
The system or method of any of claims 56-59, wherein the Cpf1 effector protein comprises a catalytically inactive RuvC domain.
The system or method of any of claims 56-60, wherein the guide RNA forms a complex with the Cpf1 effector protein and directs the complex to bind a target DNA, and wherein the cytidine deaminase converts a C to a U in the non-targeted strand of the target DNA.
The system or method of claim 65, wherein the Cpf1 effector protein is fused to a heterologous functional domain having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, or nucleic acid binding activity.
Dependencies are inferred from the claim text.